Energy balance (involving energy metabolism, nutritional state, lipid storage and the like) is an important criteria for health. This energy homeostasis involves food intake and metabolism of carbohydrates and lipids to generate energy necessary for voluntary and involuntary functions. Metabolism of proteins can lead to energy generation, but preferably leads to muscle formation or repair. Among other consequences, a lack of energy homeostasis lead to over or under formation of adipose tissue.
Formation and storage of fat is insulin-modulated. For example, insulin stimulates the transport of glucose into cells, where it is metabolized into xcex1-glycerophosphate which is used in the esterification of fatty acids to permit storage thereof as triglycerides. In addition, adipocytes (fat cells) express a specific transport protein that enhances the transfer of free fatty acids into adipocytes.
Adipocytes also secrete several proteins believed to modulate homeostatic control of glucose and lipid metabolism. These additional adipocyte-secreted proteins include adipsin, complement factors C3 and B, tumor necrosis factor xcex1, the ob gene product and Acrp30. Evidence also exists suggesting the existence of an insulin-regulated secretory pathway in adipocytes. Scherer et al., J. Biol. Chem. 270(45): 26746-9, 1995. Over or under secretion of these moieties, impacted in part by over or under formation of adipose tissue, can lead to pathological conditions associated directly or indirectly with obesity or anorexia.
Acrp30 is a 247 amino acid polypeptide that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of a amino-terminal signal sequence, a 27 amino acid stretch of no known homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and a carboxy terminal globular domain. See, Scherer et al. as described above and International Patent Application No. WO 96/39429. Acrp30, an abundant human serum protein regulated by insulin, shares structural similarity, particularly in the carboxy-terminal globular domain, to complement factor C1q and to a summer serum protein of hibernating Siberian chipmunks (Hib27). Expression of Acrp30 is induced over 100-fold during adipocyte differentiation. Acrp30 is suggested for use in modulating energy balance and in identifying adipocytes in test samples.
Another secreted protein that appears to be exclusively produced in adipocytes is apM1, described, for example, in Maeda et al., Biochem. Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone had a 244 amino acid open reading frame and a long 3xe2x80x2 untranslated region. The protein included a signal sequence, an amino-terminal non-collagenous sequence, 22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminal region with homology to collagen X, collagen VIII and complement protein C1q.
Complement factor C1q consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being about 225 amino acids long with a near amino-terminal collagen domain and a carboxy-terminal globular region. Six triple helical regions are formed by the collagen domains of the six A, six B and six C chains, forming a central region and six stalks. A globular head portion is formed by association of the globular carboxy terminal domain of an A, a B and a C chain. C1q is therefore composed of six globular heads linked via six collagen-like stalks to a central fibril region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a bouquet of flowers. Acrp30 has a similar bouquet structure formed from a single type of polypeptide chain.
C1q has been found to stimulate defense mechanisms as well as trigger the generation of toxic oxygen species that can cause tissue damage (Tenner, Behring Inst. Mitt. 93:241-53, 1993). C1q binding sites are found on platelets. Additionally complement and C1q play a role in inflammation. The complement activation is initiated by binding of C1q to immunoglobulins
Inhibitors of C1q and the complement pathway would be useful for anti-inflammatory applications, inhibition of complement activation and thrombotic activity.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect, the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 51-246 of SEQ ID NO:2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxyl-terminal C1q domain comprising 10 beta strands. Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 23-246 of SEQ ID NO:2. Within another embodiment the collagen domain consists of 15 Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats. Within another embodiment the carboxyl-terminal C1q domain comprises the sequence of SEQ ID NO:5. Within another embodiment the carboxy-terminal C1q domain comprises amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212, 217-222 and 236-241 of SEQ ID NO:2. Within another embodiment any differences between the polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within another embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within still another embodiment the polypeptide comprises residues 23-246 of SEQ ID NO:2. Within another embodiment the collagen domain consists of amino acid residues 51-113 of SEQ ID NO:2. Within yet another embodiment the C1q domain consists of amino acid residues 114-246 of SEQ ID NO:2. Within another embodiment the polypeptide is covalently linked at the amino or carboxyl terminus to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.
Also provided is an isolated polypeptide selected from the group consisting of: a) a polypeptide consisting of a sequence of amino acid residues that is 80% identical in amino acid sequence to amino acid residue 51 to amino acid residue 113 of SEQ ID NO:2, the polypeptide consisting of Gly-Xaa-Xaa and Gly-Xaa-Pro repeats forming a collagen domain; b) a polypeptide consisting of a sequence of amino acid residues that is 80% identical in amino acid sequence to amino acid residue 114 to amino acid residue 246 of SEQ ID NO:2 comprising the sequence of SEQ ID NO:5; and c) a polypeptide consisting of a sequence of amino acid residues that is 80% identical in amino acid sequence to amino acid residue 51 to 246 of SEQ ID NO:2, the polypeptide consisting of Gly-Xaa-Xaa and Gly-Xaa-Pro repeats forming a collagen domain and comprising the sequence of SEQ ID NO:5.
Within another aspect is provided a fusion protein comprising a first portion and a second portion joined by a peptide bond, the first portion consisting of a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 51 to amino acid residue 246 of SEQ ID NO:2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 246; c) a portion of the zacrp3 polypeptide of SEQ ID NO:2, comprising the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; d) a portion of the zacrp3 polypeptide of SEQ ID NO:2, comprising the C1q domain or an active portion of the C1q domain; or e) a portion of the zacrp3 polypeptide of SEQ ID NO:2 comprising of the collagen-like domain and the C1q domain; and the second portion comprising another polypeptide. Within one embodiment the first portion is selected from the group consisting of: a) a polypeptide consisting of the sequence of amino acid residue 51 to amino acid residue 113 of SEQ ID NO:2; b) a polypeptide consisting of the sequence of amino acid residue 114 to amino acid residue 246 of SEQ ID NO:2; c) a polypeptide consisting of the sequence of amino acid residue 51 to 246 of SEQ ID NO:2.
The invention also provides a polypeptide as described above; in combination with a pharmaceutically acceptable vehicle.
Within another aspect, the invention provides an antibody or antibody fragment that specifically binds to a polypeptide as described above. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b); and d) human monoclonal antibody. Within another embodiment the antibody fragment is selected from the group consisting of F(abxe2x80x2), F(ab), Fabxe2x80x2, Fab, Fv, scFv, and minimal recognition unit. Within another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described above.
Within another aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 51-246 of SEQ ID NO:2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxyl-terminal C1q domain consisting of 10 beta strands. Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 23-246 of SEQ ID NO:2. Within another embodiment the collagen domain consists of 15 Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats. Within another embodiment the carboxyl-terminal C1q domain comprises the sequence of SEQ ID NO:5. Within another embodiment the carboxy-terminal C1q domain consists of amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212, 217-222 and 236-241 of SEQ ID NO:2. Within another embodiment any differences between the polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within yet another embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within another embodiment the polypeptide comprises residues 23-246 of SEQ ID NO:2. Within another embodiment the collagen domain consists of amino acid residues 51-113 of SEQ ID NO:2. Within yet another embodiment the C1q domain consists of amino acid residues 114-246 of SEQ ID NO:2.
Also provided is an isolated polynucleotide selected from the group consisting of: a) a sequence of nucleotides from nucleotide 1 to nucleotide 1696 of SEQ ID NO:1; b) a sequence of nucleotides from nucleotide 69 to nucleotide 806 of SEQ ID NO:1; c) a sequence of nucleotides from nucleotide 135 to nucleotide 806 of SEQ ID NO:1; d) a sequence of nucleotides from nucleotide 219 to nucleotide 806 of SEQ ID NO:1; e) a sequence of nucleotides from nucleotide 408 to nucleotide 806 of SEQ ID NO:1; f) a sequence of nucleotides from nucleotide 69 to nucleotide 407 of SEQ ID NO:1; g) a sequence of nucleotides from nucleotide 135 to nucleotide 407 of SEQ ID NO:1; h) a sequence of nucleotides from nucleotide 219 to nucleotide 407 of SEQ ID NO:1; i) a polynucleotide encoding a polypeptide, the polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 51 to 113 of SEQ ID NO:2; j) a polynucleotide encoding a polypeptide, the polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 114 to 246 of SEQ ID NO:2; k) a polynucleotide encoding a polypeptide, the polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 51 to 246 of SEQ ID NO:2; l) a polynucleotide encoding a polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 23 to 113 of SEQ ID NO:2; m) a polynucleotide that remains hybridized following stringent wash conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1; n) nucleotide sequences complementary to a), b), c), d), e), f), g), h), i), j), k), l) or m) and o) degenerate nucleotide sequences of i), j), k) or l).
Also provided is an isolated polynucleotide encoding a fusion protein comprises a first portion and a second portion joined by a peptide bond, the first portion is selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residues 51 to 246 of SEQ ID NO:2; b) a polypeptide comprising the sequence of amino acid residues 1 to 246 of SEQ ID NO:2; c) a polypeptide comprising the sequence of amino acid residues 23 to 246 of SEQ ID NO:2; d) a polypeptide comprising the sequence of amino acid residues 23 to 113 of SEQ ID NO:2; e) a polypeptide comprising the sequence of amino acid residues 1 to 113 of SEQ ID NO:2; f) a portion of a polypeptide of SEQ ID NO:2 comprising the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; g) a portion of the polypeptide of SEQ ID NO:2 containing the C1q domain; or h) a portion of the polypeptide of SEQ ID NO:2 including the collagen-like domain and the C1q domain; and the second portion comprising another polypeptide.
Also provided is an isolated polynucleotide consisting of the sequence of nucleotide 1 to nucleotide 738 of SEQ ID NO:10.
Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator. Within one embodiment the DNA segment encodes a polypeptide that is at least 90% identical in amino acid sequence to residues 23-246 of SEQ ID NO:2. Within another embodiment the collagen domain consists of 15 Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats. Within another embodiment the carboxyl-terminal C1q domain comprises the sequence of SEQ ID NO:5. Within another embodiment the carboxy-terminal C1q domain consists of amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212, 217-222 and 236-241 of SEQ ID NO:2. Within another embodiment differences between the polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within yet another embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within a further embodiment the DNA segment encodes a polypeptide comprising residues 23-246 of SEQ ID NO:2. Within another embodiment the collagen domain consists of amino acid residues 51-113 of SEQ ID NO:2. Within yet another embodiment the C1q domain consists of amino acid residues 114-246 of SEQ ID NO:2. Within yet another embodiment the DNA segment encodes a polypeptide covalently linked at the amino or carboxyl terminus to an affinity tag. Within another embodiment the DNA segment further encodes a secretory signal sequence operably linked to the polypeptide. Within a related embodiment the secretory signal sequence comprises residues 1-22 of SEQ ID NO:2.
Within another aspect, the invention provides a cultured cell into which has been introduced an expression vector as described above, wherein the cell expresses the polypeptide encoded by the DNA segment.
Within another aspect, the invention provides a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector as describe above; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.